Contributions
Abstract: EP373
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
The transcription factor CCAAT-enhancer-binding protein alpha (C/EBPα) is a master regulator of granulopoiesis. Usage of two alternative translation initiation sites in the CEBPA mRNA results in expression of a full-length C/EBPα protein, termed p42 (42 kDa) and a shorter p30 isoform (30 kDa). CEBPA is mutated in 10-15 % of Acute Myeloid Leukemia (AML) patients. While N-terminal frameshifts that ablate the expression of p42 represent the predominant type of lesions, mutations in the C-terminal basic-region leucine zipper (bZip) that disrupt the DNA-binding ability of C/EBPα are also found. AML patients harbor either mono- or biallelic CEBPA mutations (CEBPAmo or CEBPAbi) and both genotypes are frequently associated with concurrent mutations in other genes. The most commonly co-occurring mutations in both groups are loss-of-function mutations in the methylcytosine dioxygenase TET2 (44.4 % in CEBPAmo / 34.8 % in CEBPAbi), resulting in adverse overall survival. We hypothesized that combinatorial effects of CEBPA mutations together with TET2 loss specifically rewire transcriptional and epigenetic circuitries in AML cells, thereby strongly influencing disease outcome.
Aims
We aimed to elucidate the molecular mechanisms behind cooperative effects of CEBPA and TET2 mutations through state-of-the-art transcriptomic and epigenomic analyses of relevant in vitro and in vivo models as well as data from AML patients.
Methods
We used the CRISPR-Cas9 technology to introduce Tet2 mutations into murine cell lines expressing only the p30 isoform (Cebpap30/p30) or mimicking biallelic CEBPA mutations (Cebpap30/C-mut.) to study functional cooperation of these mutations in vitro. A Cebpa-/p30 Tet2-/- mouse model was used to study effects of Tet2 loss in CEBPA mutated AML. We performed RNA-, C/EBPα-ChIP-, ATAC- and Bisulfite-seq to generate a comprehensive dataset for in-depth comparative analysis and correlation with relevant patient data from the beatAML collection.
Results
CRISPR/Cas9-mediated introduction of Tet2 mutations into Cebpap30/p30 and Cebpap30/C-mut. cell lines conferred a strong proliferative advantage to Tet2-targeted cells in vitro. In line, loss of Tet2 in a mouse model of CEBPA mutant AML shortened leukemia latency. TET2 loss caused decreased global chromatin accessibility and increased DNA methylation, which was accompanied by downregulated expression of numerous genes. Integration of transcriptomic and epigenomic data from in vitro and in vivo models with gene expression analyses in AML patients harboring CEBPA and TET2 mutations identified the transcription factor GATA2 as a conserved target of the CEBPA-TET2 axis. ChIP-seq showed that p30 binds to multiple regions in the Gata2 locus, and Tet2 loss caused reduced accessibility and increased DNA methylation of the Gata2 promoter. RNAi-mediated silencing revealed a dose-dependent effect of Gata2 expression on leukemia cell fitness in vivo. Furthermore, treatment with the demethylating agent 5-azacytidine restored Gata2 expression in an AML model with Cebpa and Tet2 mutations and caused a significant survival benefit.
Conclusion
The datasets generated from these novel models enable deeper insights into the global epigenetic and transcriptomic changes that depend on CEBPA and TET2 mutations in a physiologically relevant mutational context. Our results reveal deregulated GATA2 expression as a result of mutational disruption of CEBPA and TET2 in AML and show that restoration of GATA2 expression can be beneficial for AML patients with CEBPA and TET2 mutations.
Keyword(s): C/EBP, Epigenetic, GATA-2, Transcription factor
Abstract: EP373
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
The transcription factor CCAAT-enhancer-binding protein alpha (C/EBPα) is a master regulator of granulopoiesis. Usage of two alternative translation initiation sites in the CEBPA mRNA results in expression of a full-length C/EBPα protein, termed p42 (42 kDa) and a shorter p30 isoform (30 kDa). CEBPA is mutated in 10-15 % of Acute Myeloid Leukemia (AML) patients. While N-terminal frameshifts that ablate the expression of p42 represent the predominant type of lesions, mutations in the C-terminal basic-region leucine zipper (bZip) that disrupt the DNA-binding ability of C/EBPα are also found. AML patients harbor either mono- or biallelic CEBPA mutations (CEBPAmo or CEBPAbi) and both genotypes are frequently associated with concurrent mutations in other genes. The most commonly co-occurring mutations in both groups are loss-of-function mutations in the methylcytosine dioxygenase TET2 (44.4 % in CEBPAmo / 34.8 % in CEBPAbi), resulting in adverse overall survival. We hypothesized that combinatorial effects of CEBPA mutations together with TET2 loss specifically rewire transcriptional and epigenetic circuitries in AML cells, thereby strongly influencing disease outcome.
Aims
We aimed to elucidate the molecular mechanisms behind cooperative effects of CEBPA and TET2 mutations through state-of-the-art transcriptomic and epigenomic analyses of relevant in vitro and in vivo models as well as data from AML patients.
Methods
We used the CRISPR-Cas9 technology to introduce Tet2 mutations into murine cell lines expressing only the p30 isoform (Cebpap30/p30) or mimicking biallelic CEBPA mutations (Cebpap30/C-mut.) to study functional cooperation of these mutations in vitro. A Cebpa-/p30 Tet2-/- mouse model was used to study effects of Tet2 loss in CEBPA mutated AML. We performed RNA-, C/EBPα-ChIP-, ATAC- and Bisulfite-seq to generate a comprehensive dataset for in-depth comparative analysis and correlation with relevant patient data from the beatAML collection.
Results
CRISPR/Cas9-mediated introduction of Tet2 mutations into Cebpap30/p30 and Cebpap30/C-mut. cell lines conferred a strong proliferative advantage to Tet2-targeted cells in vitro. In line, loss of Tet2 in a mouse model of CEBPA mutant AML shortened leukemia latency. TET2 loss caused decreased global chromatin accessibility and increased DNA methylation, which was accompanied by downregulated expression of numerous genes. Integration of transcriptomic and epigenomic data from in vitro and in vivo models with gene expression analyses in AML patients harboring CEBPA and TET2 mutations identified the transcription factor GATA2 as a conserved target of the CEBPA-TET2 axis. ChIP-seq showed that p30 binds to multiple regions in the Gata2 locus, and Tet2 loss caused reduced accessibility and increased DNA methylation of the Gata2 promoter. RNAi-mediated silencing revealed a dose-dependent effect of Gata2 expression on leukemia cell fitness in vivo. Furthermore, treatment with the demethylating agent 5-azacytidine restored Gata2 expression in an AML model with Cebpa and Tet2 mutations and caused a significant survival benefit.
Conclusion
The datasets generated from these novel models enable deeper insights into the global epigenetic and transcriptomic changes that depend on CEBPA and TET2 mutations in a physiologically relevant mutational context. Our results reveal deregulated GATA2 expression as a result of mutational disruption of CEBPA and TET2 in AML and show that restoration of GATA2 expression can be beneficial for AML patients with CEBPA and TET2 mutations.
Keyword(s): C/EBP, Epigenetic, GATA-2, Transcription factor